standard. Since the pathlength is constant (1 cm) in the spectrophotometer, L is constant, concentration of the

 

In many laboratories, the gel-based techniques are restricted

to qualitative studies or are used as reference methods.

During the last decade, the gel techniques are increasingly being replaced by optical detection methods.

The various techniques by which quantitative

immunoassays are performed can be broadly grouped as

follows:

Heterogeneous Immunoassays

These assay systems employ an antibody immobilized on

a solid phase, which captures the corresponding antigen

from the sample. A second labeled antibody specific to a

different epitope of the antigen is used as a basis for signal

generation. After the immunochemical reaction has taken

place, the bound and unbound-labeled antibodies are

separated. The concentration of antigen is then estimated by

measuring bound or unbound-labeled antibodies through

an appropriate signal generation and measurement system.

Heterogeneous immunoassays can be performed by

various techniques such as:

¾ Radioimmunoassays (RIA)

¾ Enzyme immunoassays (EIA)

¾ Fluorescent enzyme immunoassay

¾ Chemiluminescent enzyme immunoassay.

The difficulties associated with separation of bound

and unbound-labeled antibodies, the need for dedicated

instrumentation and labor intensive procedures has

prompted the usage of heterogeneous assays in speciality

laboratories mainly through use of expensive automation.

The need for simpler, affordable, user-friendly assay

techniques for detection of routinely encountered clinical

analytes still remained to be explored. With the tremendous

progress made in instrumentation technology, optics, and

software, the face of quantitative estimation for routine

parameters has changed dramatically in the recent years.

Simultaneous development in purification techniques for

polyclonal antibodies, emergence of monoclonal antibodies

with high specificity and avidity have been instrumental in

the development of homogeneous assay techniques which

are simple to perform and easily adaptable for routine

laboratory analysis.

Homogeneous Immunoassays

These assays require only the mixing of a sample (antigen)

and the immunochemical reagents (antibody) followed by

detection of signal. These assays do not require separation

of free or bound-labeled materials in the test system for the

detection or measurement of the antigen. In homogeneous

immunoassays the immunochemical binding produces a

detectable signal (agglutination, absorbance, fluorescence,

etc.). The simplicity and flexibility associated with the

performance of homogeneous assays has made their

usage popular with laboratorians worldwide.

The homogeneous assays can be performed by different

techniques such as:

¾ Turbidimetry

¾ Nephelometry

¾ Homogeneous enzyme immunoassays

¾ Enzyme-multiplied immunoassay technique (EMIT)

¾ Enzyme inhibitor immunoassay

¾ Enzyme complementation immunoassay

¾ Substrate linked fluorescence immunoassay (SLFIA)

¾ Scintillation proximity assay (SPA)

¾ Electrochemiluminescence (ECL)

¾ Luminescent oxygen channeling immunoassay (LOCI).

The clinical chemistry analyzers (photometers) were

originally developed for colorimetric estimation of chemical

or enzymatic reactions. Subsequently, it was shown that

the visible scattered light in Kraus’s precipitin reaction

could be measured by turbidimetry and nephelometry on

photometers, to quantitate the immune complex formation.

These systems utilize the fast reaction between an antigen

with their corresponding antibodies in a liquid phase.

The technique of quantitation by turbidimetry and

nephelometry is apparently similar to the popular

absorption spectrophotometry used in routine clinical

laboratories and hence, adaptable by high throughput as

well as small and medium laboratories easily.

Spectrophotometry

Spectrophotometers work on the basis of the Beer’s and

Lambert’s law.

Beer’s Law

When a colored solution is illuminated with a

monochromatic light (light of a single wavelength), its

absorbance is proportional to the concentration of the

colored solution when the light path is constant, i.e. AαC

where A is the absorbance of light, C is the concentration

of solution.

Lambert’s Law

When a colored solution is illuminated with a

monochromatic light, its absorption is proportional to

the length of the light path, when the concentration of the

702 Concise Book of Medical Laboratory Technology: Methods and Interpretations solution is constant, i.e. A α L, Where A is the absorbance

of light, L is the length of the light path.

Beer-Lambert Law

Combining the two laws together, we have the BeerLambert law, which states that when a colored solution is

illuminated by a monochromatic light, its absorbance is

proportional to the concentration of the solution and the

length of the light path, i.e. A α C α L.

A = K × C × L .....Equation (1)

where K is a constant

In all photometric estimations a reference standard

whose concentration is known is used and its color

intensity is compared with the color intensity of the test

sample, i.e. At = K × Ct × L

As = K × Cs × L

where At

—Absorbance of test, Ct

—Concentration of test,

As—Absorbance of standard, Cs—Concentration of the

standard.

Since the pathlength is constant (1 cm) in the

spectrophotometer, L is constant, concentration of the

standard Cs is known, therefore,

 At

Ct = _____ × Cs ......Equation (2)

 As

It has been observed with most biochemistry

analytes that as the concentration of analyte increases

linearly, the absorbance also increases linearly within

the pathophysiological concentration. When a graph of

concentration vs absorbance is plotted, a straight-line graph

is obtained (Fig. 23.1A). A single standard method using a

standard of known concentration or a factor method can be

employed for calculating the concentration of the unknown.